Arylsulfonyl derivatives of pteroylglutamic acid and intermediates therefor



Patented July 31, 1951 UNITED STATES PATENT. OFFICE ARYLSULFONYL DERIVATIVES oF PTER- OYLGLUTAMIC ACID AND INTERMEDI- ATES THEREFOR "David I. Weisblat and Barney J. Magerlein, Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application July 31, 1948,

Serial No. 41,887 I e 11 Claims. (01. 26092515) a N N cu ry-Oomm momcnloohoa' Z E k wherein R is from the group consisting of hydrogen and the alkyl radicals, Z is an arylsulfonyl radical and 7'1. is an integer from the group 1 to7,

inclusive. I

In the naming'of compounds of the invention 'when both a' glutamic acid residue and a paminobenzoic acid residue are included in the molecule; the nitrogen atom of the glutamic acid residue is, for "convenience, herein referred to by the symbol N' and the nitrogen atom of the p-amino-benzoic acid residueiis referred to by the symbol N. given, compounds containing more than one 'glutamic acid or ester "residue contemplated by the inventionare those wherein only the gammacarboxyl groups are involved in the peptide linkages.

The method and intermediate compounds of the invention are of value in. the preparation of certain compounds 'ofthe group known in the art as folio acids which have been isolated from natural sources. Thus,"N'-(N((2-ami-no 4-hydroxy- 6 pteridyl)methyD p-aminobenzoyl) glutamic, acid (pteroylglutamic 'acid) wherein the glutam-ic acid residue has the same configuration as l(+)-g-lutam,icwacid and whichcan be prepared readily 1 by splitting the ip-toluenesulrfonylgroup 3 from -"N-- (N-(-(2.-amino4-hydroxy- 6 -.-pteridyl) methyl-) ep-toluenesulfonyl p arnino benzoyl) -glutamic acid, as hereinafter described,

appears to --be identical with the-so called- L. r

cc'sei factor cry-vitamin Bc isolated "from liver. Other compounds of R the same general nature, but having two or more glutamic acid residues connected through the gamma-carboxyl group which can be prepared lay-splitting the arylsul- .ionyl radicalfrom still other N'-(N-((2-amino- :4-hydroxy-6-pteridyl)-methyl) -arylsulfonyl p aminobenzoyl) -glutamicj acids or. esters appear to. beidenticalwith and to have the same bio- .l e id ii o series pa i i-emit As indicated by't-he formula 2 acid group. The value of methods and intermediates usefulpin preparing .these and related compounds syntheticallyis apparent. g

The reactions involved in the method of the invention are indicated .in the accompanying diagram. v

/\ 2 CHHP OOX III glutamic acid compound COOR' N- (N- ((2-amino-4-hyd roxy-6-pteridy1) methyl) -ary1sulfonyl-paminobenzoyl) -glutamic acid compound According to the method of the invention an N ((2 -amino- 4-hydroxy 6 pteridyhmethyllvarylsulfonyl-peaminobenzoice acid (I) is first treated with a phosphorus pentahalide to convert I it to the corresponding N-'('(2-amino-4-hydroxyr (i-pteridyl) methyl) -arylsulfonyl-p-aminobenzoyl halide (II) which is subsequently condensed with a glutamic acid compound, (III) to give the. desired N (N ((2-amino-4 hydroxy-6pteridy1) r methyl) -aryls.u1fo-nylp-aminobenzoy1) -glutamic acid compound .(IV) In the diagram, R, Z and n have the values previously mentioned and the halogen (X) can bechlorine or bromine.

The N(('2am'ino ;4 hydioxy 6 pteridyl): methyl) -arylsulfonyl jp-aminobenzoic acid used as starting material in the present process can be prepared in any convenient way, one way being by the reaction of an arylsulfonyl halide, i. e. a chloride orbromide, with N-((2-amino- 4 hydroxy-6-pteridyl)methyl) -p-aminobenzoic acid. The latter compound can be prepared readily by the method described and claimed in a co-pending application, Serial No. 32,528. According to the method of the co-pending application, 2,4,5-triamino-6-hydroxypyrimidine is first condensed with dihydroxy-acetone to form 2 amino-4-hydroxy-G-hydroxymethyl-pteridine which is then converted by treatment with a hydrogen halide, i. e. with hydrogen bromide or hydrogen chloride, to the corresponding 2-aminod-hydroxy-6halomethyl-pteridine. The latter compound is then condensed with p-aminobenzoic acid to form the N-((2 amino-4 hydroxy- S-pteridyl) methyl) -paminobenzoic acid. Other ways of preparing the N-((2-amino4-hydroxy- 6 pteridyhmethyl) arylsulfonyl p aminobenzoic acids are described and claimed in a concurrently filed co-pending application, Serial No. 41,882.

The N-((2-amino-4-hydroxy 6 pteridyl)- methyl-arylsulfonyl-p-aminobenzoic acids used in the process can contain substantially any arylsulfonyl radical with the formation of the corresponding intermediate and final products. Arylsulfonyl radicals which may be present in the starting material and in the ensuing intermediate and final products include the benzenesulfonyl, p-toluenesulfonyl, o-toluenesulfonyl and naphthalenesulionyl radicals, as well as many others.

Due to the ready availability and low cost of the p-toluenesulfonyl halides, and to the higher yields of amine sometimes obtained when splitting a p-toluenesulfonyl derivative of an amine,

as hereinafter described, than when splitting a reference to p-toluenesulfonyl compounds.

Glutamic acid compounds (III) which can be used in the method of the invention comprise glutamic acid, gamma-glutamylglutamic acid, gamma glutamyl-gamma-glutamylglutamic acid and the like having from 1 to '7 glutamic acid residues in the molecule linked through the gamma-carboxyl groups, and their alkyl esters. Although the invention'will be described in the case of esters with particular reference to the ethyl esters, it is understood that other alkylesters, such as the methyl, propyl, iso-propyl, butyl, tert.-butyl, hexyl, nonyl and dodecyl esters, can be used with equal facility, if desired.

Compounds similar to or identical with those of the folic acid group made by-using the intermediates or methods of the invention, such as pteroylglutamic acidand pteroylgamma-glutamyl-gamma-glutamylglutamic acid, which are of greatest value as measured by their biological activity-against Lactobacz'llus casez' or Streptococcus fecalis R, are those wherein the glutamic acid 4 residues possess the same configuration as l(+) glutamic acid and for this reason the preferred compounds of the invention are those having the same configuration. However, the invention also contemplates compounds having the dextro configuration as well as racemic mixtures.

The reaction of an N-((2-amino-4-hydroxy-6- pteridyDmethyl)arylsulfonyl p aminobenzoic acid with a phosphorus pentahalide is carried out conveniently by refluxing a mixture of approximately equi-molecular quantities of the two substances. Refiuxing is usually carried on for from several minutes to a few hours, during which time hydrogen halide is evolved. A phosphorous oxyhalide can be used as a diluent, if desired. The mixture is then evaporated in vacuo to remove excess phosphorus pentahalide and the phosphorus oxyhalide formed during the reaction as well as that added as a diluent. The N-((2-amino-4- hydroxy 6 pteridyDmethyl) arylsulfonylp-aminobenzoyl halide remains as an oily or solid residue which is generally sufiiciently pure for use in the second step of the process without further treatment.

The reaction of an N((2-amino-4-hydroxy-6- pteridyhmethyl)arylsulfonyl p aminobenzoyl halide with a glutamic acid compound is carried out conveniently by agitating a mixture of these substances, preferably in about equi-molecular proportions, with at least approximately the theoretical amount of dilute aqueous sodium hydroxide or other aqueous alkali necessary to combine with the hydrogen halide formed. In the case of a glutamic acid compound which is an acid, sufiicient alkali should be used to form the salt of the glutamic acid compound and in the case of glutamic acid compounds which are esters, care should be taken to avoid the use of an excess of too strong an alkali or of unduly high temperatures to prevent hydrolysis of the ester groups, unless such hydrolysis is desired. By using more vigorous reaction conditions, such ester groups can be hydrolyzed concurrently with the reaction of the acid halide with the glutamlc acid compound, if. desired.

The reaction is usually affected by agitating a mixture of the acid halide, the glutamic acid compound and the aqueous alkali at ordinary room temperatures although higher temperatures can be employed, if desired. The reaction is usually substantially complete in from about 30 minutes to about 2 hours at ordinary temperatures and proceeds somewhat more rapidly at elevated temperatures. The reaction mixture is then acidified, preferably to a pH of about 3, and the precipitate collected, washed and dried. The N'-(N( (2-amino-4-hydroxy 6 pteridyhmeth yl) -arylsulfonyl-p-aminobenzoyl) -glutamic acid compound is thus obtained as an amorphous solid which can, if desiredfbe purified by decoloriza tion and reprecipitation from dilute alkali. Esters formed by using a glutamic acid ester can be hydrolyzed to the corresponding acids with alkalies, if desired.

The splitting of an arylsulfonyl radical from an N'-(N-((2-amino 4 hydroxy 6 pteridyl) methyl) -arylsulfonyl-p-aminobenzoyl) glutamic acid compound can be accomplished according to the method described and claimed in a concurrently filed co-pending application, Serial No. 41,883. According to the method of the co-pending application, the arylsulfonylamino compound is treated, usually at ordinary temperatures, with hydrogen bromide and a bromine acceptor, such as phenol, catechol, naphthol or the like, inan aliphatic acid medium such as acetic acid. The mixture is allowed to stand for from one to several hours during which -time the arylsulfonyl radical is split from the molecule. The presence of the bromine acceptor in the reaction mixture serves to "prevent bromination in the benzene nucleus of the arylamine, such as invariably occurs during the reaction when the bromine acceptor is omitted. The hydrobromide of the amine can be isolated in high yield by pouring the reaction mixture into ether whereupon the hydrobromide separates in crystalline form. In this way, a diethyl N'-(N-((2amino-4-hydroxy-6- pteridyl) methyl) aryl sulfonyl p aminobenzoyl) -glutamate can be converted readily and in high yield to diethyl N'-(N-((2-amino-4-hydroxy- 6 -pteridyl) methyl) -p-aminobenzoyl ,-glutamate. The latter ester "can, if desired, be hydrolyzed readily to the corresponding dicarboxylic acid. Other sulfonylamino acids or esters can be converted to the respective amines in similar manner.

Certain advantages of the invention are apparent from the following examples which are given by way of illustration only and are-not to be construed as limiting.

Errample 1.--2-amz'no -4- hydroxy- :6 hydroxy .meth'yZ-pteridine One hundred twenty milligrams of dihydroxyacetone was added to a filtered solution of 100 milligrams of 2,4,-5-tria'mi-no fi hydroxypyrimidine in 15 milliliters of normal aqueous acetic acid and the solution was heated on the steam bath.

The color changed from pinkthrough yellow to orange in "the course of several minutes and crystals began to separate. A fter heating for twenty minutes the mixture was cooled and filtered. Thirty milligrams of crystalline Z-amino- 4-hydroxy-6 hydroxymethylepteridine was thus obtained. The filtrate was cooled over night in a "refrigerator and an additional 20 milligrams of the hydroxymethyl compound was obtained upon filtering. v

An additional 100 milligrams of dihydroxyacetone was ,.added to thei'lltratev from the hydroxymethyl' "compound and the mixture heated andicooled as before. .A third'cropof crystalline 2 amino 4 hydroxy -6-:hydroxymethyl pteridine was thus obtained making .a total yield of 80 milligrams. Ultra-violet absorption data showed the pteridine ring to be completely aromatized. After dissolving in dilute alkali and reprecipitating the acid, the compound was essentially pure. It decomposes at above 350C. Anal. Calcd. for C7H'IO2N5+ /gH2OZ C, 41.58; 3.99; N,.34.64; E20, 4.49 Found:

C, 40.71; H, 4.29; N, 34.90; H20, 4.18

Example 2.2-amino-4-hydr0xy-6- chloromethyl-pteridine Fifty milligrams of 2-amino-4-hydroxy-6-hydroxymethyl-pteridine was mixed with one molecular proportion of zinc chloride and one molecular proportion of concentrated aqueous hydrochloric acid. Solution occurred slowly, the final The solid consisted of 2-amino-4- hydroxy-6-chloro1i1ethy1 pteridine which was used without further purification. The bromide is prepared in similar manner.

Example .3.-N-( (.2-aminq-4-hydroary-G-pterialyl) methyl) -p-amino-beneoic acid One molecular proportion each of -2-'amino-4=-. hydroxy -6-chloromethylpteridine and of 1p aminobenzoic acid and two molecular proportions of aqueous sodium bicarbonate were mixed. and the'mixture refluxed for about four hours. At the end ofthis time the solution contained only a very smalliamount of insoluble-matter. Upon cooling thesolution and acidifying, a-precipitate of pteroic acid formed which was recovered by centrifuging and washing twice with cold water and once "with absolute ethanol. The product wasibiologically active when testedby the method of 'ileply and :Elvehjem (J. Biol. Chem. 157 303 -(.l945).)-

' 'N-r( (2-zamino -4- hydroxy -6- pteridirli-methyl) p-aminobenzoic acid is converted to 'N-'('(2 amino -4- hydroxy -6- pteridyl) methyl) -ptolu- 'enesu'lfonyl-p-aminobenzoic acid by agitation with p-toluenesulfonyl chloride and aqueous sodium hydroxide. The compound is recovered readily by extracting the mixture with ether to remove unreaoted p-toluenesulfonyl chloride,-subsequently acidifying the extracted aqueous solution to decompose the sodium salt of N-('(2- amino-l-hydroxy-6-pteridyl) methyl) p-toluenesulfonyl-p-aminobenzoic acid and filtering the mixture.

Example 4.--.N ((2 amino 4 hydroxy 6- tem'dybmethyl) p toluene sulfonyl paminobenzoic acicl Sixty-six milligrams of sodium acetate and 56.6 milligrams of 2,4,5 triamino 6 hydroxypyrimid'ine were added to a solutio'n of N-(S-hydroxy- Z-ketopropyl) p toluenesulfonyl-p-amino'benzoic acid in 9 milliliters of glacial acetic acid. The solution was allowed to stand at room temperature for one hour and was then heated for twenty minutes on a steam bath. After standing :at room temperature exposed to the air overnight, the acetic acid was distilled under reduced pressure at about 50 C. leaving a residue of N-((2- aminol-hydroxy-6-pteridy1) methyl) -p-t'oluenesulfonyl-p-aminobenzoic acid.

Example 5.N ((2 ammo 4 hydroxy 6- pteridybmethyl) p tolaenesalfonyl pamivwbenaoyl chloride A mixture of 50 milligrams of N((2-amino-4- hydroxy 6 pteridyl)methyl) p toluenesu'lfonyl-p-aminobenzoic acid, 200 milligrams of phosphorus pentachloride and 5 milliliters of phosphorus oxychloride was refluxed for minutes. The solution was then evaporated to dryness in vacuo. There was thus obtained a thick oily residue consisting of N-((2-amino-4-hydroxy 6 pteridyDmethyl) p toluenesulfonyl-p-aminobenzoyl chloride which was utilized in Example 6 without further purification. The acid bromide is prepared in similar manner using phosphorus pentabromide instead of phosphorus pentachloride.

Example 6.-N' (N ((2 amino 4 hydroxy- 6 pteridg/Dmethyl) p toluenesulfonyl pamznobeneoyl) -Z-glutamic acid A solution of 0.25 gram of l(+) -glutamic acid in 25 milliliters of 0.2 normal aqueous sodium hydroxide solution was added to the N-((2-amino 4 hydroxy 6 pteridyDmethyl) p toluene sulfonyl-p-aminobenzoyl chloride obtained in Example 5 and the mixture shaken until the solid had dissolved. After standing at room temperature for about one hour the pH was adjusted to 3.0 and the precipitate collected, washed and dried. The dried product weighed 30 milligrams and consisted of N-(N-((2-amino-4-hydroxy-6- pteridyl) methyl) -p-toluenesulfonyl-p-aminobenzoyl) -l-glutamic acid which, after removal of the p-toluene-sulfonyl radical, stimulated the growth of L. casez'. The diethyl ester is prepared in similar fashion using diethyl l-glutamate.

In similar fashion N-((2-amino-4-hydroXy-6- pteridyl) methyl) p toluenesulfonyl p aminobenzoyl chloride or bromide is reacted with gamma-glutamyl-gamma-glutamylglutamic acid or its alkyl ester to produce N'-(N-((2-aminohydroxy 6 pteridyl) methyl p toluenesulfonyl p aminobenzoyl) gamma glutarnylgamma-glutamylglutamic acid or its ester, respectively.

We claim:

1. The method which comprises agitating a mixture including an aqueous alkali, an acid halide from the group consisting of the N-((2- amino--hydroxy -6- pteridyl) methyl) arylsulfonyl-p-aminobenzoyl bromides and chlorides and a glutamic acid compound having the formula COQR' nNndnomomcoon' wherein R, is from the group consisting of hydrogen and the alkyl radicals, and separating from the reaction mixture a compound having the formula COOR OH I N T jom-N-OcoNHc/Homcmooorv z HNL 6. The method which comprises reacting an N-((2-amino- 4 -hy-droxy 6 pteridyDmethyl) arylsulfonyl-p-aminobenzoic acid with a phosphorus pentahalide from the group consisting of COOR wherein R has the values given and Z is the same arylsulfonyl radical contained in the acid halide. '7. The method of claim 6 wherein the phos phorus halide is phosphorus pentachloride.

8. The method of claim 6 wherein the arylsulfonyl radical is the p-toluenesulfonyl radical.

9. A compound having the formula XI Tom- 000): z .N

wherein X is from the group consisting of bromine and chlorine and Z is an arylsulfonyl radical.

10. N-((2-amino 4 hydroxy 6 pteridyl)- methyl) arylsulfonyl-p-aminobenzoyl chloride.

11. N-((2-amino 4 -hydroxy 6 pteridyD- methyl) -p-tolueneso1fonyl-p-aminobenzoyl chloride.

DAVID I. WEISBLAT. BARNEY J. MAGERLEIN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Hultquist etal. June 8, 1948 OTHER REFERENCES Wolf et al.: J. Am. Chem. Soc., 69, 2754 (1947). Spies et al.: Blood 3, 122 (January 1948). Lederle Bulletin, 13 (No. 3), 21 (19 :8).

Number Certificate of Correction Patent No. 2,562,223 July 31, 1951 DAVID I. WEISBLAT ET AL.

It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction as follows:

Column 3, lines 25 and 26, for pteridyl)methylread ptefidyl) methyl) column 7, line 19, for pteridyl) methylread ptem'dg bmethyl) and that the said Letters Patent should be read as corrected "above, so that the same may conform to the record of the casein the Patent Oflice.

Signed and sealed this 4th day of March, A. D. 1952.

THOMAS F. MURPHY,

Assistant Commissioner of Patents. 

6. THE METHOD WHICH COMPRISES REACTING AN N-((2-AMINO- 4 -HYDROXY - 6 -PTERIDYL) METHYL)ARYLSULFONYL-P-AMINOBENZOIC ACID WITH A PHOSPHORUS PENTAHALIDE FROM THE GROUP CONSISTING OF PHOSPHORUS PENTABROMIDE AND PHOSPHORUS PENTACHLORIDE TO FORM AN N-((2-AMINO-4-HYDROXY-6PTERDIY)METHYL)-ARYLSULFONYL - P - AMINOBENZOYL HALIDE, AGITATING A MIXTURE INCLUDING THE N-(2AMINO-4-HYDROXY-6-PTERIDYL)METHYL) - ARYLSULFONYL-P-AMINOBENZOYL HALIDE, AND AQUEOUS ALKALI AND A GLUTAMIC ACID COMPOUND HAVING THE FORMULA
 10. N-((2-AMINO - 4 - HYDROXY- 6 -PTERIDYL)METHYL)-ARYLSULFONYL-P-AMINOBENZOYL CHLORIDE. 